ABSTRACT Current treatments for obesity fail to significantly impact body weight and protect against its devastating health consequences. A promising approach to induce weight loss is to increase energy expenditure and nutrient disposal. However, the incomplete understanding of the mitochondrial mechanisms and physiological factors that regulate energy expenditure has hindered progress. Uncoupling protein-3 (UCP3) is a skeletal muscle- enriched member of the widely conserved class of mitochondrial anion / solute carrier superfamily of proteins linked in a variety of clinical genetic studies to obesity in prone human populations. UCP3 activation increases insulin sensitivity, fatty acid oxidation, and thermogenesis, and loss of UCP3 promotes obesity under high caloric load. Targeting UCP3 (and UCP1) for increasing energy expenditure, while highly promising, has been confounded by (A) the lack of understanding of how it regulates fat oxidation, (B) how it is regulated at a molecular level, and (C) what it actually transports. Work in this application addresses each of these gaps in uncoupling protein biology and importantly, significantly delves into how UCP1 functions as well. We discovered that UCP3 regulates mitochondrial C4 substrate (malate, aspartate) transport directly (when reconstituted into liposomes) and in muscle mitochondria from wild type but not UCP3 knockout mice. We also found that UCP3 forms a complex with mitochondrial malate dehydrogenase (MDH2), which converts malate to oxaloacetate, the mitochondrial metabolite necessary for complete fatty acid oxidation. Finally, we show that skeletal muscle-specific UCP3 expression rescues drug-induced thermogenesis (a response that requires fatty acids) in global UCP3 knockout mice, showing that muscle may be a novel site of UCP3 thermogenesis. The overall working hypothesis of this proposal is that UCP3 coordinates the maximal capacity for skeletal muscle thermogenesis and fat oxidation through the control of mitochondrial malate and potentially other C4 metabolite mitochondrial import (anaplerotic flux of malate and likely aspartate, among others). We will test this in the following Aims: (1) Define the role of UCP3 in C4 metabolite mitochondrial transport and the molecular mechanisms involved. (2) Examine the mechanisms and physiological relevancy of UCP3-dependent metabolite transport for fat oxidation and UCP3-mediated thermogenesis. Significance summary: This work will provide fundamental and novel insights into the molecular mechanisms regulating UCP3 (and likely UCP1), and will identify novel UCP-modulating ?druggable? targets and mechanisms.